CD47 is a widely expressed cellular receptor well known for its immunoregulatory functions. By interacting with its ligands, including thrombospondin-1 (TSP-1), signal regulatory protein α (SIRPα), integrins, and SH2-domain bearing protein tyrosine phosphatase substrate-1 (SHPS-1), it modulates cellular phagocytosis by macrophages, transmigration of neutrophils and activation of dendritic cells, T cells and B cells. Ample studies have shown that various types of cancer express high levels of CD47 to escape from the immune system. Based on this observation, CD47 is currently considered as a prominent target in cancer therapy.

Introduction of CD47

Cluster of differentiation 47 (CD47), also known as integrin-associated protein (IAP), is a 50-kDa transmembrane protein belonging to the immunoglobulin (Ig) superfamily, which is comprised of an extracellular amino-terminal Ig domain, five highly hydrophobic putative membrane-spanning segments, and a short cytoplasmic tail. It is encoded by CD47 gene, which located on 3q13.12 region of chromosome in human. CD47-SIRPα axis was considered as a tumor phagocytosis checkpoint signal, which transmits the "don't eat me" signal to macrophage and is utilized as an immune evasion mechanism by various cancers. The binding of CD47 to SIRPα induces tyrosine phosphorylation of two tyrosine residues in the intracellular immunoreceptor tyrosine-based inhibitory motif of SIRPα and recruiting the protein tyrosine phosphatases Src Homology 2 (SH2)-containing protein tyrosine phosphatase 1 (SHP-1) and SHP-2. These signaling changes result in alterations of a variety of substrates and downstream signaling pathways, including inhibition of non-muscle myosin IIA, thereby restricting the phagocytic function of macrophage. Blocking CD47 by monoclonal antibodies reactivated the phagocytosis function of macrophage and significantly decreased the tumor burden in mice models with hematologic neoplasms or solid tumors, dependent on the existing of macrophages rather than other immune cells.

Fig. 1 Regulation of phagocytosis by CD47-SIRPα axis.

Regulation of CD47 Expression

• Cytokines

Tumor necrosis factor alpha (TNF-α) is a member of the TNF/TNFR cytokine superfamily, widely involved in promotion and progression of chronic inflammatory disease and malignant tumors. TNF-α could regulate a range of cytokines in the tumor microenvironment such as CCL8, vascular endothelial growth factor (VEGF). TNF-α was reported as an inducer of CD47 expression in various types of cancer cells. Treatment with 100 ng/ml of TNF-α for 8 h led to enhancement of almost four times CD47 transcriptional expression in MCF7 breast cancer cells and the same concentration of TNF-α increase nearly four times CD47 transcriptional expression in HepG2 hepatoma cancer cells after 48 h. Upon TNF/TNFR1 pathway stimulation, NF-κB was released and entered the nucleus where it activated CD47 gene through binding to a CD47-associated specific super enhancer, which consists of multiple enhancers that are composed of a series of transcription factor proteins that bind together to drive the transcription of genes involved in cell identity. Additionally, VEGF blockade substantially enhanced TNF-α expression in relapsing non-small cell lung cancer (NSCLC) cells, leading to the enhancement of CD47 expression whereby TNF-α/NF-κB signaling pathway.

• Oncogenes

Uncontrolled tumor cell proliferation and abnormal blood vessel formation lead to the deprivation of oxygen in tumor tissue, inducing a hypoxic condition. Hypoxia in tumors is correlated with the alteration of cellular metabolism, angiogenesis, drug resistance, mainly modulated by hypoxia-inducible factors 1 (HIF-1). Meanwhile, hypoxia also induced HIF-1-dependent immune evasion mechanisms including enhancing CD47 expression in the tumor microenvironment. HIF-1 directly bound with the promoter of CD47 to activate gene transcription and increased cell surface CD47 in HCC1954 and SUM159 breast cancer cells. Moreover, macrophage phagocytosis rate was significantly increased when co-cultured with HIF-1-deficient breast cancer cells, indicating that HIF-1 plays a pivotal role in regulation of CD47 expression.

Uncontrolled tumor cell proliferation and abnormal blood vessel formation lead to the deprivation of oxygen in tumor tissue, inducing a hypoxic condition. Hypoxia in tumors is correlated with the alteration of cellular metabolism, angiogenesis, drug resistance, mainly modulated by hypoxia-inducible factors 1 (HIF-1). Meanwhile, hypoxia also induced HIF-1-dependent immune evasion mechanisms including enhancing CD47 expression in the tumor microenvironment. HIF-1 directly bound with the promoter of CD47 to activate gene transcription and increased cell surface CD47 in HCC1954 and SUM159 breast cancer cells.

• MicroRNAs

MicroRNAs are small non-coding RNAs consisting of about 20-24 nucleotides and play a pivotal role in cell proliferation, differentiation and apoptosis. MicroRNAs negatively control the expression of genes by mediating degradation of target mRNA and/or inhibiting their translation. In human cancer, the role of microRNAs in regulating CD47 expression has been revealed. miR-708, functions as a tumor suppressor, directly bound the 3′-UTR of CD47 and inhibited CD47 expression in T cell acute lymphoblastic leukemia.

• Enzymes

Various types of enzymes are involved in the regulation of a target protein synthesis. Glutaminyl-peptide cyclotransferase-like protein (QPCTL, isoQC) belongs to a family of enzymes which catalyze the formation of N-terminal glutamine and glutamic acid residues on target protein into an N-terminal pyroglutamate residue (pGlu). Notably, the presence of N-terminal pyroglutamate formation of CD47 protein, which is considered to be the post-translational modification of CD47, could be specifically recognized by SIRPα and contributed to the binding of CD47 to SIRPα.

CD47-Based Strategies to Eliminate Cancer Cells

• Peptide-based approaches

One of the best-known mimicry peptides binding to CD47 is 4N1K. 4N1Kmediated activation of CD47 has been shown to trigger caspase independent apoptosis in some human breast cancer cell lines through a Gi signaling-mediated reduction of cAMP levels and inhibition of PKA. Secretion of pro-inflammatory cytokines including IL-12, TNFα, IL-6 and GM-CSF may also be inhibited by 4N1K binding to CD47 through blocking the maturation of dendritic cells. This leads to a reduced intensity and duration of the inflammatory response.

• Antibody-based approaches

Due to the high expression of CD47 on cancer cells, the use of anti-CD47 antibodies can selectively target tumor cells for macrophage-mediated phagocytosis. Since some normal cells also express CD47, anti-CD47 antibodies may be expected to also induce phagocytosis in these cells by macrophages, but this has not been observed in practice. This observation may be due to the fact that the level of CD47 expression on cancer cell surfaces is higher than that on normal cell surfaces, and that antibodies thus tend to target cancer cells more efficiently than normal cells. Hu5F9-G4 is an anti-CD47 humanized antibody binding monomeric human CD47, thereby preventing activation of the CD47-SIRPα axis and promoting phagocytosis of tumor cells via innate immune cells. In addition, it has the ability to control the growth of different hematological malignancies in vivo. A novel CD47-blocking molecule, ALX148, has been generated by fusing the inactivated human IgG1 Fc with a modified SIRPα D1 domain. Blocking CD47 by ALX148 has been found to induce both innate and acquired immune responses against tumors. In fact, ALX148 has been shown to promote dendritic cell activation, macrophage phagocytosis, a shift of tumor-associated macrophages towards an inflammatory phenotype, an increased T cell effector function, a proinflammatory cytokine production and a reduction in the number of suppressive cells within the tumor microenvironment.

• SiRNA and miRNA-based approaches

The use of miRNAs and siRNAs to downregulate CD47 expression has been considered as an effective approach to treat cancer. In an animal model of melanoma, intravenous delivery of liposomeencapsulated anti-CD47 siRNAs was able to inhibit tumor growth. In addition, the migration of colon cancer cells was decreased in the presence of M2 macrophages by CD47-targeting siRNAs. However, some challenges intrinsic to gene silencing strategies (i.e., siRNAs and miRNAs) may limit their therapeutic application. Specifically, their low bioavailability and poor cellular uptake may result in a suboptimal delivery to target tissues as well as in off-target effects and, thus, to safety concerns.

CD47 Related Products & Applications

Creative Biogene is committed to providing customers with innovative products to explore the functions and applications of CD47. Our products include transfected stable cell lines, preformed viral particles and cell lysates, providing comprehensive solutions for studying CD47-related processes.

• Transfected stable cell lines

Our transfected stable cell lines express CD47 or its variants, providing a reliable and consistent cellular model for investigating CD47 function, signaling pathways, and drug responses. These cell lines serve as invaluable tools for studying the role of CD47 in immune regulation, cancer biology, cellular signaling, and other relevant research areas.

• Preformed viral particles

Our preformed viral particles, such as lentiviral or adenoviral particles, contain CD47 genes or shRNA constructs designed to modulate CD47 expression. These particles offer an efficient and targeted approach to deliver CD47-related genetic material into specific cell types. Researchers can utilize these particles to study CD47 function, assess its role in disease progression, or explore potential therapeutic interventions.

• Cell lysates

Our CD47 cell lysates provide a valuable resource for researchers to study the protein expression, post-translational modifications, and protein-protein interactions involving CD47. These lysates are derived from cells expressing CD47, enabling researchers to investigate CD47-related mechanisms and protein interactions using techniques such as Western blotting or immunoprecipitation.